Guanine nucleotide binding proteins (G proteins) are ubiquitous proteins that transduce signals from ion channels, growth factors, hormone and neurotransmitters to a variety of intracellular effectors. In-vitro reconstitution studies with purified receptor, G protein and effector suggest that several different G proteins can interact with receptors and effectors. Other mechanisms play a role in-vivo to restrict the range of possible interactions. One way this may be achieved is by localizing the G protein to a specific plasma membrane domain. Many cells including epithelial cells segregate plasma membrane proteins (including some G proteins) to distinct surfaces. The broad long term objectives of this proposal are to define the mechanisms utilized by epithelial cells to localize G protein subunits to specific plasma membrane domains and to define the structural requirements of alpha subunits necessary for specific membrane targeting. The goals are to develop a cell culture model for the study of specific membrane localization. alphai2 localizes basolaterally in both MDCK and LLC-PK1 cells and will be labeled with an epitope from an unrelated protein to distinguish transfected from endogenous subunits. In-vitro characterization of the tagged alphai2 subunit will allow alpha subunit properties including interaction with beta gamma, binding of GTP, and myristoylation to be studied. Epitope tagged alpha i2 will be characterized to determine conditions for immunoprecipitation by the anti-epitope antibody. Tagged alpha i2 will be localized in cells by immunofluoresence and Western blots. Structural features of alpha necessary for proper sorting will be defined using mutations in alpha i2 based on my previous in-vitro work with alpha omicron. Amino and carboxyl terminal mutations in tagged alpha i2 and, chimeric alpha i2 proteins will be made to define these regions. The timing and pathway(s) of sorting will be defined using pulse chase labelling experiments and selective membrane purification. Immunoprecipitation studies using purified vesicles will begin to define other cellular elements involved in the sorting process. G proteins are critical to many cellular processes, and changes in G proteins have been linked to several diseases including cholera and Albright's Hereditary Osteodystrophy. Activating mutations in alpha/ s and alpha/i have been described in several human endocrine neoplasms. Polar distribution of plasma membrane proteins is altered in many disease states. Defining mechanisms of G protein subunit localization will further our understanding of cellular responses in normal and diseased states.